Coagulation of blood is a complex process requiring the sequential interaction of a large number of components, nearly all of which are proteins. These components include fibrinogen and Factors II, V, VII, VIII, IX, X, XI, and XII. A lack of any of these components, or a nonfunctional component, can lead to an inability of the blood to clot when required, with resultant excessive and life-threatening blood loss to the patient.
Factor VIII is absent or is present at deficient levels in certain individuals. For example, persons who have a deficiency (or absence) of Factor VIII, i.e., persons suffering from hemophilia A, have blood which either fails to clot or clots only after longer periods of time than the time required for clotting in a person who has a normal level of Factor VIII.
Factor VIII is present in plasma as a high-molecular-weight complex (Factor VIII complex), which includes Factor VIII:C and von Willebrand factor (Factor VIII:RAg or vWf). Factor VIII:C promotes blood coagulation. Factor VIII:RAg interacts with platelets to promote aggregation of the platelets and, when incorporated in the Factor VIII complex, acts as a stabilizer for Factor VIII:C.
The primary therapeutic use of Factor VIII has been its intravenous administration to hemophilia A patients. In severe cases, relatively high concentrations of Factor VIII are required. These high concentrations are obtained by purification and concentration of Factor VIII. However, purification often leads to instability and loss of Factor VIII:C activity because of the removal of Factor VIII:RAg from the Factor VIII complex during purification. Thus, the resultant purified product is often a mixture of both stable Factor VIII complex and unstable Factor VIII:C, along with contaminating proteins that have not been removed.
Some processes for producing Factor VIII concentrate have been based on a discovery by Poole et al. (Nature, 203, p. 312 (1964)) that the precipitate remaining after plasma is frozen and then thawed, i.e., the cryoprecipitate, contains Factor VIII in a concentrated form and excludes various other protein fractions. It was discovered that, in addition to Factor VIII, the cryoprecipitate also includes the major portion of the fibronectin component of plasma.
Work progressed over the years to perfect the separation of Factor VIII from other proteins in the cryoprecipitate, including the fibronectin component, so that the resultant products would incorporate increased concentrations of Factor VIII relative to the other proteins present in the plasma.
One area in which a substantial amount of work has been done in the production of Factor VIII is based on the use of polyethylene glycol (PEG) to precipitate Factor VIII from other proteins in an aqueous cryoprecipitate solution. For example, U.S. Pat. No. 3,652,530, which issued on Mar. 28, 1972 to A. J. Johnson, discloses a process for preparing a Factor VIII concentrate by fractionating cryoprecipitate with PEG at a relatively lower concentration to precipitate fibrinogen and other proteins, and then increasing the PEG concentration to precipitate Factor VIII.
U.S. Pat. No. Re. 29,698 to Fekete et al. discloses a process for production of Factor VIII by which heparin is added to a cryoprecipitate solution, along with PEG, to provide increased yields of precipitated Factor VIII. It is disclosed that the amount of heparin employed during the fractionation step can vary, with the optimum concentration being one unit of heparin per ml of the plasma solution, whereas concentrations of heparin greater than about 10 units per ml are to be avoided as dangerous.
Purification of Factor VIII by chromatography on heparin (European Patent Application. No. 90308104.0 to Battacharya et al. and Madras et al., Haemostasis, 7, 321-331 (1978)) has also been used. The Madras process, while producing Factor VIII complex, results in a Factor VIII complex with little or no activity. The Battacharya process results in an active Factor VIII; however, the specific activity of the protein purified was at best 66 units/mg. (The phrase "specific activity" as used herein means units of Factor VIII:C clotting activity per milligram of protein. A "unit" is defined as the amount of Factor VIII:C in one ml of normal plasma.)
Purification of Factor VIII has also been achieved by chromatography on monoclonal antibody-containing chromatography media (U.S. Pat. Nos. 4,361,509 and Re. 32,001 to Zimmerman et al.). Such procedures result in Factor VIII:C of very high specific activities, approximately 1500 to 2500 units/mg of total protein, and, therefore, high purity. However, in the Zimmerman process, the Factor VIII:RAg is dissociated from Factor VIII:C, which results in the Factor VIII:C being unstable.
Currently, the methods used to purify Factor VIII result in protein preparations, i.e., Factor VIII concentrates, which have a relatively low Factor VIII:C specific activity. It is desirable that there be provided an improved process for the separation of Factor VIII complex, i.e., the intact Factor VIII:C/Factor VIII:RAg complex, from contaminating proteins for producing Factor VIII which results in increased purity, higher concentration, and enhanced yields and stability of the protein.